Genotyped functional screening of soluble Fab clones enables in-depth analysis of mutation effects

Monoclonal antibodies (mAbs) and their fragments are widely used in therapeutics, diagnostics and basic research. Although display methods such as phage display offer high-throughput, affinities of individual antibodies need to be accurately measured in soluble format. We have developed a screening platform capable of providing genotyped functional data from a total of 9216 soluble, individual antigen binding fragment (Fab) clones by employing next-generation sequencing (NGS) with hierarchical indexing. Full-length, paired variable domain sequences (VL–VH) are linked to functional screening data, enabling in-depth analysis of mutation effects. The platform was applied to four phage display-selected scFv/Fab screening projects and one site-saturation VH affinity maturation project. Genotyped functional screening simultaneously enabled the identification of affinity improving mutations in the VH domain of Fab 49A3 recognizing Dengue virus non-structural protein 1 (NS1) serotype 2 and informed on VH residue positions which cannot be changed from wild-type without decreasing the affinity. Genotype-based identification revealed to us the extent of intraclonal signal variance inherent to single point screening data, a phenomenon often overlooked in the field. Moreover, genotyped screening eliminated the redundant selection of identical genotypes for further study and provided a new analysis tool to evaluate the success of phage display selections and remaining clonal diversity in the screened repertoires.

selection) for 1 h, collecting the beads with DynaMag-2 bar magnet and transferring the supernatant to the positive panning reaction.
The third round of selection was performed by providing the antigen, anti-digoxigeninscFv-SpyCatcher clone 203A2 (Department of Life Technologies, University of Turku) in solution at 2.5 nM concentration and mixed with 1 x 10 10 cfu 2nd round phages in 2 mL assay buffer RED. The binding reaction was incubated for 30 min at RT on rotation, after which, 50 µg of paramagnetic M280 streptavidin Dynabeads (Invitrogen, USA) precoatedto saturation with biotin-S-S-digoxigenin conjugate (Department of Life Technologies, University of Turku) were added to the reaction. The incubation was continued for 30 min on rotation followed by washes, elution and phage rescue as above. The following day, a DNA miniprep was prepared from the cell mass harbouring the 3rd round output pEB32x-scFv phagemids.

Conversion of scFv output library into Fab input library by selective RCA
The enriched anti-SpyCatcher scFvs from the 3rd round were converted into Fab format with selective RCA 2 . A VL-CL-VH -mega primer was prepared by amplifying VL and VH domain genes from the 3rd round pEB32x-scFv anti-SpyCatcher miniprep DNA with primer pairs HS012/WO375 and HS015/HS034 using Phusion II HSDNA polymerase (Thermo, USA) according to manufacturer´s instructions, respectively. Similarly, CL domain was amplified using codon-harmonized Fab construct pEB32x hrFab0 miniprep DNA 3 as the template with primers EB197 andHS014. All PCR reactions were amplified with the thermal cycling protocol: (1) init.den. at 98°C for 30 s, (2) den. at 98°C for 5 s, (3) ann. at. 60°C for 15 s, (4) ext. at 72°C for 25 s (a total of 25 cycles, steps 2-4) and (5) final ext. at 72°C for 5 min. The PCR products were purified with GeneJet PCR purification kit (Thermo, USA) and 500 ng of purified products were digested with 10 U of LguI in 20 µl reaction in 1x Tango bufferovernight at 37°C.
The LguI reactions were purified as above and a triple fragment ligation was set up containing 50 ng of each of the LguI digested fragments, 1 x T4 DNA ligase buffer and5 U of T4 DNA ligase (Thermo, USA) in a total volume of 20 µl. The ligation was incubated overnight at 16°C followed by two consecutive PCR reactions to amplify the VL-CL-VH fragments. The first PCR reaction contained 5 µl sample of the ligation as template in a total reaction volume of 200 µl using primers WO375 and HS034. The PCR reaction was purified as above and a 2.5 µl sample was taken as the template for the second asymmetric PCR reaction performed in a 100 µl volume. The latter reaction contained only primer WO375. Both reactions were thermally cycled as above with Phusion II HSDNA polymerase (except for 30 cycles instead of 25). The PCR reactions were purified with GeneJet PCR purification kit and 750 ng of the ssDNA PCR product (contains a mix of ss and dsDNA) was 5´-phosphorylated in a 30 µl reaction volume containing 1 x T4 PNK buffer and 15 U of T4 polynucleotide kinase (Thermo, USA). The reaction was incubated overnight at 37°C and stored frozen at -20°C for further use. Selective RCA was performed as described earlier 2 . The final product, pEB32x-Fab anti-SpyCatcher library, was transformed to SS320 cells by electroporation. Illustration of the panning scheme can be seen in Figure S15.

Selection of anti-SpyCatcher Fab clones by phage display
A Fab display phage stock was prepared from the SS320 cells harbouring pEB32x-Fab anti-SpyCatcher phagemid DNA with VCS-M13 (Stratagene, USA) superinfection according to standard protocol. The Fab-phages were selected against in vivo biotinylated SpyCatcher (produced in BL21/ BirA+ E. coli strain and purified in Department of Life Technologies, University of Turku) and SdyCatcher.
First round of selection against in vivo biotinylated SpyCatcher and SdyCatcher proteins were performed in parallel. First, a negative selection step was done by preincubating 5 x 10 10 cfu phages from the generated stock with 100 µg of M280 streptavidin Dynabeads for 1 h, collecting the beads with DynaMag-2 bar magnet and transferring half of the supernatant to the positive panning reaction (other half used as control reaction with no antigen on beads). The positive selections were done by incubating collected phages with 100 µg of M280 strepavidin Dynabeads saturated with the biotinylated Catchers in1 mL of TBT-0.05 (50 mM Tris, 150 mM NaCl, 1 % BSA, 0.05 % Tween-20, pH 7.5).The binding reactions were incubated for 1 h at RT on rotation, washed two times on bar magnet with TBT-0.05 and once with TBS (50 mM Tris, 150 mM NaCl, pH 7.5), eluted with trypsin and rescued as above. DNA minipreps were extracted from scraped XL-1 Blue cell mass (GeneJet Plasmid miniprep kit, Thermo, USA) harbouring enriched pEB32x-Fab anti-SpyCatcher and anti-SdyCatcher repertoires.
On second selection round, 30 µg of neutravidin coated Dynabeads (Department of Life Technologies, University of Turku) saturated with in vivo biotinylated Catchers were incubated with 1 x 1010 phages from 1st round Fab-phage stocks in 1 mL of TBT-0.05. The binding reactions were incubated for 1 h at RT on rotation, washed twice on bar magnet with TBT-0.1 (50 mM Tris, 150 mM NaCl, 0.1 % Tween-20, pH 7.5) and once with TBS, eluted with trypsin and rescued as above. DNA minipreps were extracted from scraped XL-1 Blue cell mass (GeneJet Plasmid miniprep kit, Thermo, USA).
Third round of selections against in vivo biotinylated SpyCatcher and SdyCatcher were performed in parallel by saturating M280 streptavidin Dynabeads with biotinylated Catchers (50 µg beads / binding reaction) and providing 5 x 10 10 cfu phages from the 2nd round Fab-phage stocks in 1 ml of assay buffer RED. The binding reactions were incubated for 30 min at RT on rotation, washed on bar magnet, eluted with trypsin and rescued as above. DNA minipreps were extracted from scraped 2/32 XL-1 Blue cell mass(GeneJet Plasmid miniprep kit, Thermo, USA) harbouring enriched pEB32x-Fab anti-SpyCatcher and anti-SdyCatcher repertoires and cloned with SfiI to pAK400 vector for screening. 10 + 10 plates were screened from both Fab libraries.

Preparation of anti-NS1 affinity maturation library for screening
For affinity maturation of anti-NS1 Fab 49A3, total of 20 sublibraries were generated using site-saturation mutagenesis PCR ( Figure S16 a). For this purpose, total 20 forward NNK-primers, each targeting single codon either in CDRH2 or CDRH3, were designed and ordered from Sigma Aldrich, USA. Mutated Fab fragments ("A") were generated with PCR with NNK-primers and pAKrev2. Non-mutated Fab fragments ("B"), containing the rest of the template Fab sequence and ovelapping sequence with corresponding fragment A, were amplified using WO375 and one of fragment B primers (Sigma), designed earlier for another project. Both fragment A and B PCR reactions consisted of 10 ng of template plasmid DNA, 0.02 U/µL Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, USA), 1 x HF Buffer, 200 µM dNTP, and 0.5 µM primers and thermal cycling was 30 cycles of 98°C (30s), 57°C (30s) and 72°C (35s) with a 98°C (30s) initial denaturing at the beginning and 72°C (5min) final elongation step at the end. The amplification was verified with gel electrophoresis on 1 % agarose gel and purified from cut gel slabs using GeneJet Gel extraction kit (Thermo Fisher Scientific). Fab cassettes of sublibraries were then assembled via PCR by mixing A and B fragments in 1:1 molar ratio and amplifying them with WO375 and pAKrev2 primers. The PCR protocol for assembly was the same as before, except of increasing annealing temperature to 58°C. The used primer sequences and fragment pairs are shown in Table S1. The used site-saturation mutagenesis PCR is illustrated in Figure  S16 b. Casettes were then cloned into pAK400 expression vector for screening by SfiI (Thermo) digestion of casette and vector, followed by ligation with T4 DNA ligase (Thermo), according to manufacturers protocol.

Preparation of anti-DARPin Fab library for screening
Anti-DARPin Fab generation was started by enriching DARPin binding antibodies in scFv format from mixed scFv M and P libraries according to standard protocol for three selection rounds 1 . The targets for the selection were biotinylated anti-lysozyme DARPin clones 378 (1st and 2nd rnd) and 398 (3rd rnd), both containing three 33 aa diversity repeats between N-and C-cap repeat. First two rounds were performed by coating DARPins to saturation on paramagnetic M280 streptavidin beads (Invitrogen) and the third round was performed by providing 1 nM and 100 pM biotinylated DARPin in solution and capturing the phage-DARPin complexes on paramagnetic streptavidin beads.
Phagemid DNA pEB32x-scFv was extracted from output cells with a miniprep kit (GeneJET, Thermo) and used as template to amplify VL and VH domains by PCR. The resulting amplicons were digested with LguI and joined to CL domain containing compatible cohesive ends with T4 DNA ligase (Thermo). The VL-CL-(RBS-PelB-)VH fragment was PCR amplified from the ligation and joined to preamplified and gel extracted (GeneJET, Thermo) CH1 domain fragment by assembly PCR. The resulting Fab fragment was amplified by PCR and cloned with SfiI (Thermo) sites to pEB32x vector for Fab selections. Phusion DNA polymerase was used in all PCR reactions according to manufacturers instructions and list of oligos used for the construction are listed in Table S2.
Fab Phage stock was prepared by transforming DNA pEB32x-Fab to SS320 cells by electroporation and preparing phage display stock from 20 mL culture according to standard protocol. Fab phage display selections were carried out as above for scFv selections for rounds one and two. Third round was performed by incubating Fab phage in microtiter wells coated with DARPin anti-GFP 3G86.32 4 fused to SNAP-tag. Final 4th round of selection was performed by providing in solution 10 nM and 1 nM biotinylated DARPin-SpyCatcher, in which the N-terminal DARPin was a polyclonal sample from a binary library containing Tyr or Ser at selected paratope positions 5 . The antigen-phage complexes were captured from buffer on paramagnetic streptavidin beads for washing and elution. The fourth round output was cloned to pAK400 vector with SfiI sites for indexing and single Fab clone screening.
Shuffling of Fab sequences to create more DNA diversity was performed between rounds 1 and 2 by performing staggered extension PCR process (StEP) 6 . StEP was performed using FirePol DNA polymerase (Solis BioDyne), 3 ng/µl of phagemid DNA in 100 µl reactions with primers WO375 and TH366. Different extension times and temperatures were used in parallel for shuffling. Fab fragments amplified in all four PCR reactions performed in different conditions were pooled after gel extraction, SfiI digested and cloned back to pEB32x with SfiI sites for 2nd round of Fab phage display selection. Illustration of the panning scheme can be seen in Figure S17.

Selection of anti-NP scFv clones by phage display
The anti-NP Fab molecules were generated in the Department of Life Technologies at the University of Turku by phage display. Synthetic antibody phage display libraries ScFvM and ScFvP 1 were selected according to standard protocol for four rounds against biotinylated SARS CoV-2 Nucleocapsid Protein with GST-tag. Antigen was kindly provided by P. Jalkanen and P.Kolehmainen from prof. Julkunen's group (Department of Virology, University of Turku), expressed in Sf9 insect cells and 3/32 purified using the GST tag. Biotinylation was done with EZ-Link NHS-PEG 4 -Biotin with 12 x molar excess of biotin, according to the manufacturers protocol. Assay buffer RED (Kaivogen Oy, Finland; 50 mmol/L Tris-HCl pH 7.75; 150 mmol/LNaCl; 0.05 % NaN3; 0.01 % Tween-40; 0.05 % bovine gamma-globulin; 20 µm/L diethylenetriamine penta-asetate; 0.5 % bovine serum albumin; 20 mg/L cherry red, later referred as AB) was used as panning buffer. Three separate phage display selections were conducted in parallel, each with different additives in AB ; triton-BSA ("N1"), triton-saliva ("N2") or no additives ("N3"). All binding reactions were washed with TBT-0.5 (50 mM Tris, 150 mM NaCl, pH 7.5, 0.5% Tween-20) using DynaMag-2 bar magnet (Invitrogen, USA) to collect the beads. Trypsin (Sigma Aldrich, USA) was used for the phage elution and XL-1 Blue cells (Stratagene, USA) for phage rescue as described earlier 1 .
On the first round of panning, 5 x 10 12 cfu of scFv-phage particles (1:1 molar mix of scFvM and scFvP libraries) were incubated for 1 h at RT on rotation with 500 µg of paramagnetic M280 streptavidin Dynabeads precoated to saturation with bio-N-GST (Invitrogen, USA; coating performed according to manufacturer´s instructions) in 2mL AB.
The second round of panning was performed similarly to the first round, except that 1 x 10 11 cfu of scFv-phages from the 1st round were used as the library input and smaller amount of streptavidin beads were used (50 µg). Also, a negative selection step was included by preincubating the scFv-phage libraries with non-coated streptavidin beads (equivalent amount of beads as for positive selection) for 1 h, collecting the beads with DynaMag-2 bar magnet and transferring the supernatant to the positive panning reaction.
The third and fourth round of selection were performed by providing the antigen in solution at 1 nM concentration and mixed with 1 x 10 10 cfu 2nd or 3rd round phages in 2 mL AB. The binding reaction was incubated for 30 min at RT on rotation, after which, 50 µg of paramagnetic M270 Epoxy beads (Invitrogen, USA), coated with Avidin (Department of Life Technologies, University of Turku) were added to the reaction. The incubation was continued for 10 min on rotation followed by five washes, elution and phage rescue as above. After fourth panning round, DNA minipreps were prepared from the cell mass harbouring the 4th round output pEB32x-scFv phagemids.

Fab conversion of panned anti-NP scFv libraries
The enriched anti-NP scFvs from the 4th round were converted into Fab format with a modified version of FASTR-method 7 . All of the used primer sequences can be found from Tables S2 and S1. Briefly, VL, CL, VH and CH1 domains of antibody were amplified with primers with LguI recognition site in overhang. VL and VH domains were amplified from the 4th round pEB32x-scFv anti-NP minipreps with primer pairs WO375/HS012 and HS015/HS016 using FIREPol DNA polymerase (Solis Biodyne, Estonia) according to manufacturer´s instructions, respectively. Similarly, CL and CH1 domains were amplified using Fab construct 001OHB12 pAK400-Fab miniprep, originating from hrFab0 3 as the template with primers pairs EB197/HS014 and EB197/pAKrev2, respectively. All PCR reactions consisted of 50 ng of template plasmid DNA, 0.05 U/µL FIREPol DNA polymerase, 1 x BD Buffer, 250 µM dNTP, and 0.1 µM primers and thermal cycling was 35 cycles of 95°C (30s), 60°C (30s) and 72°C (30s) with a 95°C (5min) initial denaturing at the beginning and 72°C (4min) final elongation step at the end. The PCR products were purified with Zymo DNA Clean & Concentrator-5 kit (Zymo Research, USA) and 400 ng of purified products were digested with 10 U of LguI (Thermo, USA) in 20 µl reaction in 1x Tango bufferovernight at 36°C.
The LguI reactions were purified as above and a four fragment ligation was set up containing 20 ng of each of the LguI digested fragments, 1 x T4 DNA ligase buffer and 5 U of T4 DNA ligase (Thermo, USA) in a total volume of 20 µl. The ligation was incubated at room temperature for one hour. The ligation products were used directly for PCR with end primers WO375/pAKrev2 to amplify the complete Fab casette. Correct amplificatin was verified by running a small amount of PCR products on 1 % agarose gel at 90 V for 40 minutes. PCR products were again purified with Zymo kit as above, followed with digestion with SfiI (Thermo) in 20 µL reaction consisting of 1 x buffer G, 20 U of SfiI and 2 µg of Fab library DNA. Digestion was incubated for 2 hours at 50°C, followed by gel extraction, after running the digestion products on 1 % agarose gel at 80 V for 40 minutes, using GeneJet Gel extraction kit (Thermo) to remove any background. The digested products were cloned into pEB32x vector to using T4 DNA ligase, same as above. The libraries in display vectors were transformed to XL1 cells by electroporation.

Selection of anti-NP Fab clones by phage display
A Fab display phage stocks for the three separate libraries were prepared from the XL1 cells harbouring pEB32x-Fab anti-NP phagemid DNA with VCS-M13 (Stratagene, USA) superinfection according to standard protocol. The Fab-phages were again selected against in biotinylated SARS-CoV-2-N. No additives were added to AB for any of the three libraries in Fab phage selections.
In first round of selection against in biotinylated N-GST a negative selection step was included by preincubating 20 µL of phages from the generated stock with mixture of M280 streptavidin Dynabeads, M270 Epoxy Dynabeads and M270 Neutravidin Dynabeads (beads from Invitrogen, USA, coated at Department of Life Technologies, University of Turku), 100 µg of each, for 1 h, collecting the beads with DynaMag-2 bar magnet and transferring half of the supernatant to the positive panning reaction (other half used as control reaction with no antigen on beads). The positive selections were done by incubating collected phages

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with 100 µg of M280 strepavidin Dynabeads saturated with the biotinylated antigen in 1 mL of AB. The binding reactions were incubated for 30 min at RT on rotation, washed two times on bar magnet while transferring beads to new tube between washes, eluted with trypsin and rescued as above. DNA minipreps were extracted from scraped XL-1 Blue cell mass (GeneJet Plasmid miniprep kit, Thermo, USA) harbouring enriched anti-NP repertoires.
On second selection round, more stringent panning was done by shorter incubation times, increased number of washes and lower concentration of antigen in solution. In addition, blocking with free GST was included to decrease the amount of GST-specific binder enrichment. 10 µL of phages from 1st round were incubated in 1 mL of AB with 100 pM bio-N-GST and 1 nM free GST and incubated for 15 minutes RT in rotation. Antigen bound phages were captured by adding 100 µg of M280 Streptavidin Dynabeads to the reaction, incubating 10 min RT, rotation and washing the beads with TBT-0.5 five times on magnet, transfering beads to new tubes between washes. Also, control reaction without antigen was included with similar steps. Elution with trypsin, rescue and minipreps were done as above.
For "NP-2" screening project, the Fab libraries from round 1 and 2 were cloned to pAK400 expression vector using SfiI restriction enzyme and T4 DNA ligase and transformed into XL1 cells for screening, as described earlier.
For "NP-1" screening project, a capture Fab for sandwich panning was screened from round 2 libraries cloned into pLK06H (Fab fusion with alkaline phosphatase) expression vector in XL1 cells, plated on agar plates supplemented with 1 % D-glucose and 100 µg of ampicillin. Briefly, 90 clones from each of the three 2nd round libraries were inoculated to 200 µL of SB (30 g/L Tryptone, 20 g/L Yeast extract, 10 g/L MOPS) supplemented with 1 % D-glucose, 100 µg of ampicillin and 10 µg of tetracyclin on 96-well plate with 3 x wells with control anti-N Fab CR3009 8 (gene purchased from Twist Bioscience, USA, produced in XL1 similarily as described in this article) and 3 x empty control wells and incubated at 37°C, 900 rpm o/n. Next morning, cells from primary culture were inoculated with four dips with 96-well replicator into fresh 200 µL of SB with 0.05 % D-glucose and antibiotics, incubated for four hours at 37°C, 900 rpm. Protein production was induced by addition of IPTG to final concentration of 200 µM, after which temperature was expression was allowed at 26°C, 900 rpm for 16 hours. Cells from original culture plates were stored at -70°C in 16 % glycerol. Cells from expression were lysed by addition of 20 µL of 10 x lysis buffer (10 mg/mL lysozyme from chicken egg white L6876 (Sigma Aldrich, United Kingdom), 25 U/µL Pierce Universal Nuclease (Thermo Fisher Scientific, USA) in PBS (20 mM sodium phosphate, 300 mM sodium chloride, pH 7.4), followed by 30 min incubation in room temperature in slow agitation, and finally freezing at -70°C.
All dilutions for immunassays were done using Assay Buffer Red and washes using Kaivogen Wash buffer, both purchased from Kaivogen. Delfia Plate Wash (Wallac, Turku Finland) instrument was used for plate washing. All incubations were done at room temperature with slow shaking. Lysate was clarified by centrifugation before the immunoassay. Clear streptavidin plates (Kaivogen Oy, Finland) were prewashed, followed by addition of 100 µL bio-N-GST with concentrations of 0, 0.3, 3, 6, 12 and 120 nM in AB and incubation of 30 minutes. Wells were washed 4 times and lysate diluted 1:5 in AB was added to each well. After 1 hour of incubation, wells were again washed 4 times, followed by addition of 100 µL pNPP buffer (1mg/mL pNPP, 500 mM Tris, 200 mM NaCl, 10 mM MgCl 2 , pH 9.0). Absorbance at 405 nm was measured after 1 hour incubation using Victor 1420 Multilabel Counter (Wallac, Finland). As there was no real signal saturation for any of the clones, 12 clones with highest specific absorbance were selected for EC 50 immunoassays. For the assay, expression of these clones was done similarily as before, but instead of 200 µL volume, on 5 mL with shaking speed of 300 rpm, inoculated from glycerol preps made earlier. Fab concentration was measured from clarified lysates diluted 1:5 to pNPP buffer with NiNTA and size exclusion chromatography purified CR3009 Fab-AP standard series.
To compare the apparent affinities against of selected clones, immunoassay on clear streptavidin plates were again carried out. 1 nM bio-N-GST was added on prewashed wells and incubated for 30 minutes, followed with 4 washes. 10 mM Fab-AP from lysates were added on wells, in addition to two controls for each clone with either 100 nM free GST (to see whether the Fab binding is dependent on the tag) or well without antigen to measure background. After 30 min incubation the wells were again washed 4 times, followed by addition of pNPP buffer and incubation of 30 min, after which absorbance at 405 nM was measured with Victor 1420 Multilabel Counter. Based on the the immunoassay results, clone from round 2 N1 library ("N1G1) was selected as capture. For this purpose, the Fab gene was cloned in pAK400 expression vector, transformed back into XL1 and expressed in 1 L culture with similar protocol as described above. Purification of the his-tagged Fab was done first with HisPur NiNTA Resin according to manufacturers protocols, and then further purified with size exclusion chromatography instrument Äkta Explorer using HiLoad 26/60 FastFlow Superdex 200 column (GE Healthcare, USA). To use the Fab as capture in panning and immunoassays, biotinylation was done using EZ-Link NHS-PEG 4 -Biotin with 20 x molar excess of biotin, according to the manufacturers protocol.
First round of panning was done with Streptavidin M280 Dynabeads saturated with bio-N1G1 capture Fab. 10 µL of round 0 phages were added to 1 mL of AB with 1 nM N-GST with presence of 10 nM free GST as blocker and incubated for 20 min, rt in rotation. Control reactions were included, similarily to above. 100 µg of capture beads were added and reaction incubated 6/32 for another 20 minutes rt, rotation, followed by 3 x washes with TBT-0.5 and once with TBS (50 mM Tris, 150 mM NaCl, pH7.5) on magnet while changing to new tubes. Elution with trypsin, rescue and minipreps were done as above.
Second round of panning was done on solid phase without presence of blocker GST. 100 µg of Streptavidin M280 Dynabeads were first saturated with bio-N1G1 capture Fab by 30 min incubation rt, rotation, washed 3 times, followed by saturation with N-GST with similar incubation and washing. 10 µL of round 1 phages were added to the reaction, followed similar incubation and washes as on round 1. Elution with trypsin, rescue and minipreps were done as above. Fab genes were cloned into pLK06H expression vector for screening, similarily as done for "NP-2". Due to complexity of the panning process, the panning is illustrated in Figure S18.

Control Fab sequences
The amino acid sequences of the Fabs used as positive controls are are shown below in Table S3. All clones share the same constant domain sequences (CH1 and CL). S001E03 was used in anti-SpyCatcher project and was discovered during initial testing of the screening platform. 248G12 was used in anti-DARPin screening, and was discovered in during earlier, unpublished project at University of Turku. N1G1 was the control Fab for anti-SARS-CoV2 projects, discovered similarly to S001E03, during the initial testing of the screening assays.             Triton-X + saliva and N3 without additives. After 4 rounds of scFv-phage selections, genes were converted into Fab format, followed with 2 more rounds of panning either similarily as in earlier rounds, or as a sandwitch with discovered N1G1 Fab bound first on magnetic beads. "R" in the name indicates the round number of the phage library.

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Processing of sequencing reads This section describes the pipeline used to process the raw FASTQ files into usable, single-clone level sequences. The pipeline includes quality analysis, merging of paired-end reads, demultiplexing, trimming adapter sequences, and obtaining unique read counts and the main sequences for each index location. We also provide descriptions of the UNIX and Python scripts used in this process. Arrows at the beginning of a code row indicate that it is part of the previous line's code. The first level of demultiplexing was performed automatically by the Illumina software after sequencing, resulting in separate FASTQ files for all screening plates. For the automatic demultiplexing step, a sample sheet was provided to the instrument, which included sample names and their corresponding TruSeq indexes.

Quality analysis of FASTQ files
FASTQC v0.11.9 was utilized to assess the quality of individual FASTQ files before and after merging the paired-end reads using UNIX commands in Ubuntu 16. A list of Illumina universal and index adapter sequences was provided to the software via the 'adapter_list.txt' file. The following code was used to generate quality reports for the raw sequencing files: fastqc raw_sequences/ * -o quality/fastqc_raw -a tables/adapter_list.txt -t 4 The reports of individual FASTQ files were then combined into single report using MultiQC v1.11 with following UNIX command: multiqc quality/fastqc_raw -i "SO_Fab_Seq raw FASTQ quality report" -o quality/multiqc_raw/ -n raw_multiqc_report Parameters used with PEAR were: -v minimum required overlap for merging, 25 bases -m maximum read length after merging, 600 bases -n minimum read length after merging, 300 bases -q minimum mean quality score of 2 subsequent bases -trim sequence after quality lower than 20 -y RAM allocation, 4 Gb -j threats used, use 6 cpu threats After merging, the read quality was again analyzed as shown in previous subsection.